| Result: 1.We successfully packaged lentivirus,infection of BRL cells,screened out the best effect of the lentivirus group,and established the IRE1 silenced stable cell lines.2.Medical fat milk culture cells induced 24 h cell fat steatosis.Oil red O staining showed the accumulation of vacuoles in the liver cells and the suggestion of cell steatosis.With oil red O staining,and CCK-8 detection of OD value,we can determine the best induction concentration of 4% of the medical fat milk.3.Morphological changes of BRL cells were determined after modeling NC: BRL cell morphology was normal at four time points(0,12,24,48h),intercellular binding was close,no red lipid droplets were found in the cells.SO group and LV-CON group: 12 h culture morphology of normal liver cells,intercellular tight combination,a little intracellular lipid droplet;cultured 24 h cells began to appear contour changes,the cell binding under tight,fat droplets increased;48h culture,cell swelling,intracellular red lipid droplets increased gradually.Group LV-IRE1: 12 h cells were cultured with normal morphology,cell tightly,some intracellular lipid droplet;morphological change of 48 h cells,24 h cells,swelling,binding under closely,intracellular lipid droplets were aggregated.4.Changes of biochemical indexes of cell culture supernatant: between the four groups and the group within three time points(12h,24 h,48h)ALT and AST were significantly different.LDH at two time points(24h,48h)between groups and within groups were statistically significant(P<0.05),while there were no significant difference in ALP,GGT(P> 0.05);TBIL,DBIL,AST,ALT and TG were significantly higher in SO group,LV-CON group and LV-IRE1 group at the three time points than that in NC group.Along with the time extending increased,the differences were statistically significant(P<0.05)for this group at three time points of 12 h,24h,48 h.LDH in 24,48 h were significantlyhigher than NC group(P <0.05).The index for group LV-IRE1 AST,ALT,LDH at three time points was significantly higher than that of SO group and LV-CON group(P<0.05),and 48 h ALB was significantly lower than the other three groups(P<0.05).5.Expression of IRE1?/XBP1s/JNK and m RNA in the four groups of BRL cells 5.1 Dynamic expression of IRE1 alpha m RNA in hepatocytes and four groups at different time points: There were significant differences in the expression of IRE1,P<0.05 and m RNA between the four groups at different time points(12,24 and 48h).There was no significant difference in the expressions of IRE1 and m RNA between the SO group and the LV-CON group at different time points(P>0.05,).Compared with the NC group,the expression of SO group and LV-CON group in 12,24,48 h time points IRE1 alpha m RNA increased significantly(P<0.05),and the expression of LV-IRE1 alpha IRE1 alpha m RNA group decreased(P<0.05);there were significant differences of expression of SO group and LV-CON group IRE1 alpha m RNA in the two groups at different time points among all increased since 12 h.It reached the peak at 24h(P<0.05),and decreased at 48 h,but no significant difference was found between 24h(P>0.05).5.2 Dynamic expression of XBP1 s and m RNA at different time points and four groups of hepatocytes: The expressions of XBP1 s and m RNA in liver cells at different time points(12,24 and 48h)were significantly different between the four groups(P<0.05).There was no significant difference in the expressions of XBP1 s and m RNA between the two groups and the LV-CON group at the different time points(P>0.05,SO).Compared with NC group,SO group and LV-CON group,XBP1 s m RNA expression in 12,24,three 48 h time points were significantly increased(P<0.05),XBP1 s m RNA LV-IRE1 a group of three time points were lower than those in group NC(P<0.05).There was no difference between group NC and group LV-IRE1 alpha at three time points(P>0.05).SO group and LV-CON group increased since 12 h,peaked at 24 h,and the index decreased at 48 h with statistical significance(P<0.05).5.3 Dynamic expression of JNK and m RNA in BRL cells and four groups: The expressions of JNK and m RNA at the three time points(12h,24 h,48h)of the four groups were statistically significant(P<0.05).There was no significant difference in the expression of JNK and m RNA between SO and LV-CON groups at different time points(P>0.05)between the two groups.Compared with the NC group,the expression was not statistically significant in SO group and LV-CON group at12h(P>0.05).At 24 h and 48 h,this two time points,JNK m RNA expression was significantly increased(P<0.05);LV-IRE1 at 12 h and 24 h in JNK m RNA alpha group was lower than that of group NC,the difference was statistically significant(P<0.05).It was significantly higher at 48 h than that of NC group(P<0.05).These three groups,SO group,LV-CON group,and LV-IRE1 group increased gradually at 12 h,and reached the peak at 48 h.The difference was statistically significant(P<0.05).6.Comparison of the expression levels of P-IRE1?/XBP1s/JNK and in hepatocytes of 24 h : 6.1 Comparison of the expression level of and P-IRE1 alpha liver cells of the four groups in 24 h protein: 24 h cells p-IRE1 protein expression in the difference between the four groups was statistically significant(P<0.05).Compared with NC group,SO group,LV-CON group,and P-IRE1 alpha protein expression increased significantly and the difference was statistically significant(P<0.05).LV-IRE1 alpha group significantly decreased(P<0.05),while SO group and LV-CON group had no significant difference here(P>0.05).Compared with the LV-IRE1 Alpha Group,SO group and LV-CON group of P-IRE1 protein expression was significantly increased,the difference was statistically significant(P<0.05).6.2 The expression levels of XBP1 s in liver cells of 24 h four groups were compared: the expression of XBP1 s protein in 24 h cells was significantly different between the four groups(P<0.05).Compared with NC group,XBP1 s protein in SO group,LV-CON group was significant higher than that in NC group(P<0.05)but there was no significant difference between the two groups,SO group and LV-CON group(P>0.05).The LV-IRE1 XBP1 s was higher than that in NC group.XBP1 s protein was increased by LV-IRE1 alpha SO group,LV-CON group.6.3 Comparison of the expression level of liver cells in rats of the four groups in 24 h JNK and P-JNK 24 h protein: the expression of p-JNK protein in JNK cells,in four groups had no statistical difference(P<0.05).JNK and P-JNK were not significantly different in protein 22(P>0.05),but it was higher than that of group NC,and the difference was statistically significant(P<0.05).Conclusion:1.This experiment successfully packaged IRE1 interference lentivirus,and successfully infected BRL cells.2.The rat BRL cells were induced by different concentrations of fat emulsion,and the optimal concentration of milk fat was selected,which laid the foundation for further experimental study.3.This experiment successfully established a model of liver cell steatosis associated with parenteral nutrition related liver disease,which laid a foundation for further study of the pathogenesis of liver fibrosis.4,SO group of liver cells of IRE1 alpha in the /XBP1s/JNK signaling pathway related molecules IRE1 alpha and XBP1 s m RNA expression in 12 h and 24 h increased,and decreased in 48 h.JNK expression of m RNA along with the time extending gradually increased.The expression of IRE1 and P-IRE1 alpha,XBP1 u,XBP1s protein in 24 h group increased;expression of LV-IRE1 related protein IRE1 alpha,XBP1 s m RNA and IRE1 and P-IRE1 alpha,XBP1 u and XBP1 s protein decreased significantly in 12,24 h,JNK m RNA and P-JNK protein were significantly increased in 24 h.When prompted by ERS,the IRE1 alpha /XBP1 s /JNK signaling pathway may be involved in the development and progression of hepatocyte steatosis in PNALD. |
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